DE69003168T2 - Isolation and structure determination of the cytostatic linear depsipeptides dolastatin 13 and dehydrodolastatin 13. - Google Patents

Abstract

The Indian Ocean sea hare Dolabella auricularia has been found to contain a new cell growth inhibitory and antineoplastic (P388 leukemia) cyclodepsipeptide designated dolastatin 13 and a companion substance dehydrodolastatin 13. A series of high field (400 MHz) 2D-NMR experiments including sequential analyses by HMBC and NOE techniques and tandem mass spectrometry provided structural determinations. Dolastatin 13 and dehydrodolastatin 13 represent a new class of cyclodepsipeptides. Pharmaceutical preparations and therapeutic regimens employing these new cytostatic depsipeptides are disclosed. The depsipeptides have the following structure: <CHEM> o

Description

The present invention relates to cytostatic linear depsipeptides designated as "dolastatin 13" and "dehydrodolastatin 13" obtained from the shell-less mollusc Dolabella auricularia found in the Indian Ocean, pharmaceutical preparations containing dolastatin 13 and dehydrodolastatin 13, respectively, as an essential active ingredient, and methods of using such preparations to inhibit cell growth in a patient suffering therefrom.

The great Roman naturalist Gaius Plinius Secundus (Pliny the Elder) described for the first time in his comprehensive study around 60 BC a highly effective sea hare of the genus Dolabella found in the Indian Ocean. (The Romans initially referred to mollusks of the family Aplysidae as sea hares because of the similarity between the ears of a hare and the ear-shaped tentacles of these gastropods). However, considerations regarding the potential uses of Indian Ocean Dolabella for modern medical problems are of recent origin (see US Pat. Nos. 4,414,205 of November 8, 1983, Dolastatine 1-3; 4,486,414 of December 4, 1984, Dolastatine A and B; and 4,816,444 of March 28, 1989, Dolastatin 10 to Pettit).

The dolastatins may correspond to the active D. auricularia components (cf. 1969, Ph.D. dissertation by M. Watson. U. of Hawaii, "Some Aspects of the Pharmacology, Chemistry and Biology of the Midgut Gland Toxins of Some Hawaiian Sea Hares, especially Dolabella auricularia and Aplysia pulmonica", University Microfilms Inc., Ann Arbor, MI.)

For centuries, the biological properties of Dolabella auricularia have been "hunted", but it was not until 1972 that this laboratory found specimens of this captivating sea hare in the Indian Ocean, which yielded extracts that were shown to be effective (over 100% increase in lifespan) against the National Cancer Institute (NCI) P388 murine lymphocytic leukemia (PS system). This laboratory subsequently succeeded in isolating ten new (and effective) cell growth inhibitory and/or antineoplastic peptides, which were designated dolastatins 1 to 10. The nomenclature is based on the source of the substance and not on any similarity in chemical structure.

From the early work, dolastatin 1 was found to be the most active (lowest dose) antineoplastic substance known at the time (33% cure rate in NCI mice B16 melanoma at 11 ug/kg). Because of the effectiveness of dolastatin, the lumpfish evidently only needs vanishingly small amounts (about 1 mg per 100 kg), which made the isolation and structural elucidation of these peptides an extraordinary challenge. Later, another substance was isolated and identified as an exceptional linear pentapeptide, designated "dolastatin 10". This substance was the most important antineoplastic component of Dolabella auricularia discovered, as it evidently represents the most active (lowest dose) antineoplastic substance found up to that time. For example, dolastatin 10 showed a 17 to 67% curative response at 3.25 to 26 ug/mg against externally grafted NCI human melanoma (nude mouse), a 42 to 138% prolongation of life at 1.44 to 11.1 g/kg using a B16 melanoma and a 69 to 102% prolongation of life at 1 to 4 ug/mg against PS leukemia (ED₅₀ = 4.6 x 10⁻⁵ ug/ml). Now dolastatin 13, a significantly different substance, has proven to be highly effective against NCI-P388 lymphocytic leukemia (PS system) (cf. Schmidt et al. "Experienta" 1978, 34, 659 - 660) - cell line with an ED₅₀ of 0.0013 ug/ml. The PS system allows an excellent activity prediction against the various types of human neoplasms (cf. Vendetti et al. "Lloydia" 30, 332 ff (1967) and the literature citations listed therein). Dehydrodolastatin 13 has similar properties.

The present invention is based on the discovery of a new and effective cytostatic substance designated "Dolastatin 13" which is extracted from the Indian Ocean shell-less mollusc Dolabella auricularia in the manner described in detail below. A related substance, namely Dehydrodolastatin 13, is also described. The substance and the related substance, their synthetic counterparts and the non-toxic, pharmaceutically acceptable derivatives thereof can be formulated with pharmaceutically acceptable carriers into valuable pharmaceutical preparations or drugs having demonstrable and confirmable rates of cell growth inhibitory activity as determined according to generally accepted protocols in use at the National Cancer Institute of the United States.

A primary object of the present invention is to provide new means for retarding or inhibiting the growth of one or more types of malignant cells.

Another object of the present invention is to develop methods and means for isolating cell growth inhibitors from marine organisms in a form in which they can be readily and usefully used in the therapeutic treatment and control of one or more types of neoplasms occurring in human patients.

A further object of the present invention is to provide means and measures for developing useful pharmaceutical preparations or medicaments for treating and controlling a neoplastic disease, these preparations or medicaments having as their essential active ingredient is a unique cytostatic factor obtained from the shell-less mollusc Dolabella auricularia found in the Indian Ocean, its synthetic counterpart or a non-toxic pharmaceutically active derivative thereof.

These and other objects are - as the following explanations show - easily achieved by the present invention in a highly unexpected manner. This follows without difficulty from the following detailed description of an exemplary embodiment of the same.

The organism

Taxonomy: Dolabella auricularia belongs to the family Aplysidae, the class Gastropoda and the order Mollusca. In a reference by H. Engel in "Zoologische Mededeelingen" Leiden, 24, 197 to 239 (1945) there are numerous color plates of Dolabella specimens. In this reference there is also a list of Dolabella species that were previously thought to be different, but which later turned out to be the same and were identified as Dolabella auricularia. These species are Dolabella agassizi, D. andersonii, D. hasseltii, D. hemprichii, D. neira, D. peronii, D. rumphii, D. teremidi, D. tongana, D. truncata, D. variegata and D. scapula.

In appearance, the Dolabella used in this case were olive-green in color with a peach-shaped body and an average length of 15 to 20 cm. The reference by H. Engel contains detailed descriptions of Dolabella collected around the world.

The Dolabella collection site used for the initial isolation of the dolastatins was located on the east side of Mauritius in the Indian Ocean, approximate location 21 south latitude, 56 east longitude, in 4 to 5 feet of water downland from the island.

Another location where Dolabella can be collected is near Negros Island in the Philippines, approximate location 9 north latitude, 123 east longitude. Extracts of Dolabella species from five separate collections all showed neoplastic activity.

Isolation and cleaning

For the isolation and purification of dolastatin 13 and dehydrodolastatin 13 from lumpfish samples, various methods can be used, e.g. solvent extraction, partition chromatography, silica gel chromatography, preparative thin layer chromatography and crystallization from solvents.

Isolation of dolastatin 13

A combined ethanol/2-propanol extract of D. auricularia (1000 kg wet weight, collected in 1982) was concentrated to an active methylene chloride fraction in a series of solvent partitioning steps. Extensive column chromatographic separation (steric digestion and partitioning on Sephadex, partitioning and adsorption on silica gel, and HPLC) using a gradient elution technique guided by a PS bioassay resulted in 25.2 mg of pure dolastatin 13 (2 x 10-7% yield from 1000 kg wet lumpfish).

A pure sample of dolastatin 13 was obtained in crystalline form from methylene chloride/hexane (10.6 mg, 6 x 10-8 % yield): mp 286-289 °C; [α]D+94 ° (c = 0.01, CH3OH); Rf: 0.56 in 90:10:0.8 CH2Cl2-CH3OH-H2O; see Scheme 1 for the mass spectrum; UV(CH3OH) λmax(logε), 220 (3.04) nm; and IR (NaCl plate): &nu;max 3384, 3315, 2960, 2930, 1733, 1677, 1653, 1529, 1205, 750 and 700 cm-1.

Based on the results of a detailed high-field (400 MHz) ¹H- and ¹³C-NMR and a high-resolution SP-SIMS peak comparison, the molecular formula C₄₆H₆₃H�7;O₁₂ was derived for dehydrodolastatin 13. A combination of ¹H, ¹H-COSY, ¹H, ¹³C-COSY and ¹H,¹H- Relay-controlled COSY experiments revealed eight discrete spin-coupled systems, four of which correspond to the known amino acids threonine (Thr), N-methylphenylalanine (MePhe) and valine (Val, 2 units). Threonine and the two valine units were also detected by amino acid analyses of the products from an acid-catalyzed (6N HCl, 110ºC, 24h) hydrolysis. An assignment of the 5th and 6th units as an N,N-disubstituted phenylalanine was realized by NMR interpretations. From a series of double and triple relay-controlled coherence transfer experiments (homonuclear relay) and sensitivity-enhanced heteronuclear multiple bond correlation experiments (HMBC), the latter Phe derivative was proven to be the new cyclic hemiacetal 3-amino-6-hydroxy-2-piperidone (Ahp), which presumably originates from a Phe-Glu dipeptide precursor (Glu-γ-carboxylaldehyde).

Continuation of the NMR experiments led to the assignment of the 7th unit as the rare dehydroamino acid α,β-dehydro-2-aminobutanoic acid (cis-Abu), presumably arising from dehydrated Thr, and the 8th (unit) as 2-O-methylglyceric acid (MeGlc). Due to some ambiguity, the Abu olefin was tentatively assigned the Z configuration shown. Confirmation of the Abu and MeGlc assignments was obtained from the results of the HMBC experiment. Initial attempts to sequence dolastatin 13 using NOE data proved unsuccessful, presumably due to a folded solution conformation and the small size of the observed NOEs. The correct sequence of the units was determined by HMBC and by combining the results from experiments in two different solvents (see Table 1 below). When dichloromethane-d₂ was used, the sequence was 1:1. Several segments of dolastatin 13 were identified as solvents due to overlapping signals in the However, for the carbonyl region, it was necessary to use data obtained in pyridine-d5, where only the chemical shifts of two carbonyls overlap. The only postulate made to complete the overall structure was that the carbonyl group at 174.38 ppm corresponds to an ester and is therefore attached to the Thr oxygen. The proposed structure shown immediately below was confirmed by sequence determination and tandem mass spectroscopy shown in Scheme 1. This structure for dolastatin 13 is as follows:

where R₁ = OH and R₂ = H.

Isolation of dehydrodolastatin 13

A small (1.72 g) barely PS-active fraction was prepared from 1,600 kg (wet weight) of D. auricularia collected in the Indian Ocean (East Africa). The fraction was further separated by gradient HPLC (RP8 silica gel, 1:1 methanol-water 100% methanol as mobile phase) (PS bioassay) to yield the previously reported dolastatin 13.

From the same fraction, dehydrodolastatin 13 was subsequently obtained as a minor component together with dolastatin 13. Crystals from methylene chloride-hexane (0.74 mg, 4 x 10⊃min;⊃9; % yield): mp 127-132 °C; [α]D +38 ° (c = 0.005, CH₃OH), Rf: 0.64 (in previous solvent system); HRSP-SIMS [M+H]+ 888.4492, calcd 888.4508 for C₄₆H₆₂N₇O₁₁; UV (CH₃OH) λmax (log ε), 220 (3.11) nm; and IR (NaCl plate): ν max 3382, 3311, 2960, 2930, 1732, 1678, 1653, 1530, 1467, 1202, 750 and 700 cm¹.

Based on the results of a detailed high-field (400 MHz) 1H and 13C NMR study and a high-resolution SP-SIMS peak comparison, the molecular formula C46H61N7O11 was derived for dehydrodolastatin 13. A combination of 1H, 1H-COSY, 1H, 13C-COSY and 1H, 1H-relay-controlled COSY (cf. Bax et al. in "J. Magn. Res." 61, 306 to 320 (1985)) experiments was also used to determine the structure of dehydrodolastatin 13 in the same way as described for dolastatin 13.

Once the structure of dolastatin 13 was established, it was clear from combined NMR and mass spectroscopy studies that dehydrodolastatin 13 was the Ahp dehydration product of dolastatin 13 and had the structure shown below. The 1H and 13C NMR spectra of the two depsipeptides appeared to be almost identical except for the Ahp signals and the amide proton of Val-2 at 7.42 (H-4), which is shifted upfield by 1 ppm upon dehydration. The latter shift indicates a hydrogen bond between H-4 and O-15 in dolastatin 13. The structure elucidated for dehydrodolastatin 13 is as follows:

where R₁ = R₂ = Δ14,15. Table 1: 1H and 13C NMR assignments and selected NOEs for dolastatin 13 in CD₂Cl₂ solution and HMBC correlations from CD₂Cl₂ and C₅D₅N solutions.* * where no multiplicity is given, it could not be determined due to overlapping signals. #Chemical shift values are interchangeable.

For a better understanding of the present invention, a more detailed description of the measures carried out in the experiments follows.

General procedures

The solvents used for chromatographic procedures were redistilled. Sephadex LH-20 (25 to 100 u) used for gel penetration and partition chromatography was obtained from Pharmacia Fine Chemicals AB, Uppsala, Sweden. For chromatographic fractionation experiments, UV-visible Gilson FC-220 track and FC-80 microfractionators connected to holochrome detectors were used. For column chromatography with silica gel, columns prepacked with 70 to 230 mesh or silica gel 60 from E. Merck (Darmstadt) were used. For HPLC, a Partisil M9 10/50 ODS-2 (C-18 reversed phase) column (9.4 mm i.d. x 500 mm) from Whatman, Inc. Clifton, NJ was used. Preparative layer plates were those from Whatman, Inc. The silica gel GF uniplates for thin layer chromatography were supplied by Analtech, Inc., Newark, Delaware. The thin layer chromatography plates were viewed under UV light and developed with an anisaldehyde/acetic acid/sulfuric acid spray (heating for 10 minutes at about 150ºC) or with ceric sulfate/sulfuric acid (heating for 10 minutes).

Amino acid analyses were performed using a Beckman Model 121. UV spectra were recorded using a Hewlett-Packard 8450A UV/VIS spectrophotometer equipped with an HP7225A plotter. IR spectra were recorded using a Nicolet MX-1 FT instrument. High resolution SP-SIMS mass spectra were recorded using VG Analytical MM ZAB-2F and Kratos MS-50 triple analysis mass spectrometers. High resolution electron impact mass spectra (m/m 10,000) were recorded on Kratos MS-80 and MS-50 instruments along with CAD spectra. Gas chromatography/mass spectrometry (GC-MS) of appropriate derivatives was performed using a J&W fused silica DB-5 column (0.243 mm x 30 m). Sequential GC-MS procedures employed chemical ionization (m/m 1,000, reagent NH3), low-resolution (m/m 1,000) and high-resolution (m/m 3,000) electron impact techniques. NMR experiments (in various solvents using a Bruker 5-mm ¹H ¹³C dual switchable probe head) were performed using a Bruker AM-400 fine-bore spectrometer with an ASPEKT 3000 computer and a oscillator operating at 400.13 and 100.62 MHz for ¹H and ¹C, respectively. ¹³C-NMR working pulse per programming unit.

Collecting the animals, extraction and preliminary tests:

The western Indian Ocean lumpfish (Mauritius), Dolabella auricularia, was first collected in October 1972. In March 1975, the activity of an ethanol extract against the National Cancer Institute (NCI) P388 lymphocytic leukemia (PS system) was confirmed with T/C 235 at 600 mg to 167 at 176 mg/kg. A series of analogous extracts from later collections of the lumpfish gave comparable results. The experiments reported here were carried out on a collection re-taken from the same site in 1982, preserved in ethanol. The total volume of animal material (1,000 kg) and ethanol preserve was 700 gallons.

After extraction and solvent partitioning, 2.75 kg of methylene chloride concentrate was obtained for large-scale preparative HPLC. Two columns in series (6" x 10') were packed with silica gel (Davisil 633, 200 to 400 mesh, packed as a slurry in 7:3 hexane/ethyl acetate). The 2.75 kg of dark (green-black) concentrate was dissolved in ethyl acetate (2 gal), pumped onto the column, and chromatographed at a rate of 60 to 72 L/h using the following solvent gradients. Eluent Eluent volume (1) Fraction No. Fraction residue (g) Hexane:Ethyl acetate Ethyl acetate Ethyl acetate-Methanol-Water

Each fraction was eluted with 20 liters of solvent. Fractions comparable (by thin layer chromatography) were pooled.

Isolation of dolastatin 13:

Of the preparative HPLC fractions, two showed significant activity in the P388 system, namely fraction A (132.0 g PS T/C toxic T 165 at 30 T 7.5 mg/kg and ED₅₀ 10⁻²) and fraction B (72.0 g, PS T/C toxic T 141 at 35 T 8.7 mg/kg and ED₅₀ 10⁻²). The fractions were combined and dried to yield 190.4 g. The major portion (152 g) was worked up as shown in the following separation scheme Part 1, Part 2 and Part 3. Separation scheme - Part 1 Fractions A (38 g) Series 1 toxic Sephadex Silica gel Separation scheme - Part 2 combined fractions K, L, M (2.6 g) Silica gel toxic same steps as for V and W Dolastatin Sephadex Hexane Separation scheme - Part 3 Fraction Sephadex Hexane Silica gel Partisil Dolastatin Separation scheme - Part 4 Fraction Sephadex Silicagel toxic Separation scheme - Part 5 combined fractions j and k (1.27 g) Sephadex hexane Partisil as for the P series Hexane Hexane-Toluene Dolastatin

Isolation and cleaning

As already mentioned, a variety of procedures can be used to isolate and purify dolastatin 13 and dehydrodolastatin 13 from lumpfish samples.

According to a preferred embodiment of the present invention, a 38 g sample of fractions A+B was chromatographed on a Sephadex LH-20 column (10 x 120 cm) in 1:1 methylene chloride-methanol. A combination of similar fractions yielded fractions C-3 as shown in the above separation scheme - Part 1. The (in vivo) active fractions D and E were combined and divided into two equal portions (6.0 each) for separation by silica gel column chromatography. Series I-1 was further separated to active fractions K, L and M by dry column chromatography with a gradient of 990:10:0.1 to 100:100:1 ethyl acetate-methanol-water. The parallel I-2 series was also separated by dry column chromatography using methylene chloride-methanol and the gradient 99:1 to 1:1 to the active fraction N. The combined fractions K, L and M (2.6 g) were separated using dry column silica gel chromatography and a 99:1 to 1:1 methylene chloride-methanol gradient - according to the above separation scheme - part 2 to obtain active fractions O-S (1.15 g). The combined active fractions O-S were then separated again on a silica gel column using a 99:1 to 4:1 methylene chloride-methanol gradient to give 10 fractions (f1Tf10). Fraction f6 (155.8 mg) was further separated on a (wet) silica gel column using 99:1 to 1:1 ethyl acetate-methanol, yielding active fractions V and W in addition to fraction f6-1.

After combining V and W (7.4 mg), a final separation was carried out in three steps including a preparative thin layer chromatography with 9:1:0.8 methylene chloride-methanol-water and again with 9:1:0.1 ethyl acetate-methanol-water with subsequent partition chromatography using SEPHADEX LH-20 and 5:5:1 hexane-methylene chloride as solvent. Thus, series I-1 yielded 5.0 mg of dolastatin 10.

On the other hand, fraction f5 (33.8 mg) (Part 2) was chromatographed with silica gel using 99:1 to 4:1 ethyl acetate-methanol-water (water: 0.1%) to obtain fraction f5-2 (13 mg; ED₅₀ 2.2 x10⁻¹). This fraction was combined with fraction f6-1 (20.9 mg; ED₅₀ 1.6 x10⁻³) after thin layer chromatography analysis. The combined fractions f5-2 and f6-1 were finally separated in three steps according to the procedures given in Separation Scheme - Part 5 for the separation of dolastatin 10, yielding 6.4 mg of crystalline dolastatin 13 (ED50 1.3 x10-2).

Additional dolastatin 13 was obtained starting with the I-2 series in Part 1. Continuation of the separation of the I-2 series afforded fraction N-1 (0.359 g). LH-20 SEPHADEX partition separation with the 4:5:1 hexane-methylene chloride-methanol solvent system of fraction N-1 afforded 2 active fractions f2 and f3 as shown in Part 3. The combined fraction (93 mg) was chromatographed on a silica gel column using 99:1→4:1 CH2Cl2-CH3OH as solvent to afford active fraction f2 (17.5 mg). Reversed phase HPLC separation (on PARTISIL-10, ODS-2) using 1:1T9:1 CH3OH-H2O afforded 9.2 mg of crystalline dolastatin 13.

The larger amount of fraction A+B (152 g) was chromatographed on columns (10x120 cm) of SEPHADEX LH-20 in five parts in 1:1 methylene chloride-methanol (see above separation scheme parts 1-3). The active fractions were combined and further separated using a column (4.5 x 80 cm; 1.2 kg) of silica gel and a step gradient of methylene chloride-methanol (49:1, 23:2, 9:1, 22:3, 17:3, 4:1, 1:1 and finally 100% methanol) to give an active fraction b (6.87 g). Fraction b was chromatographed on silica gel (dry) using a 99:1 to 1:1 methylene chloride-methanol gradient. The resulting active fractions d- i (4.6 g) were combined and chromatographed on a (dry) silica gel column using a 99:1 to 1:1 ethyl acetate-methanol gradient to give active fractions j and k (1.27 g).

Two fractions (j and k combined) were chromatographed on SEPHADEX LH-20 using a 5:5:1 hexane-methylene chloride-methanol partition system to give an active fraction m. Separation of fraction m on silica gel (size B Merck prepacked) using a 99:1 to 1:1 methylene chloride-methanol gradient gave active fractions O-1 (58.8 mg), O-2 (20.6 mg) and O (91.6 mg), respectively.

At this point, fraction O was used in two parts. Fractions p and q were separately purified in parallel using preparative thin layer chromatography (90:10:1 ethyl acetate-methanol-water as mobile phase) and subsequent successive SEPHADEX LH-20 partition steps with 5:1:1 hexane-methylene chloride-methanol, 5:5:1 hexane-ethyl acetate-methanol and finally with the 5:5:1 hexane-toluene-methanol solvent system. Fraction p yielded 8.6 mg and fraction q 11.3 mg of pure dolastatin 10: Total yield: 28.7 mg of an amorphous (colorless) powder (mp 107-112º) from methylene chloride-methanol.

The active fraction O-2 (20.6 mg; ED50 1.5 x10-2) was separated by reverse phase HPLC (ODS-2) with 1:1→9:1 CH3OH- H2O to yield 9.6 mg of crystalline dolastatin 13.

Separation of the highly active fraction on silica gel (size B, Merck, prepacked) using a 99:1 to 1:1 methylene chloride-methanol gradient regime afforded an active fraction f4 (12.8 mg; ED50 7.2 x 10-4) as shown in Separation Scheme, Part 3. The resulting active fraction was finally purified by HPLC (silica gel ODS-2 column) using a 1:1 to 9:1 methanol Water gradients. This procedure yielded 6.2 mg of pure dolastatin 13.

The structure of dolastatin 13 was given on page 7. The spectral and HMBC correlations are given in Table I on page 9.

Dolastatin 13, the related dehydrodolastatin 13, their synthetic counterparts and their pharmaceutically active physiologically acceptable derivatives can be administered in a highly effective manner to animals or humans suffering from a neoplastic disease associated with malignant cell growth, for example acute myelocytic leukemia, acute lymphocytic leukemia, malignant melanoma, adenocarcinoma of the lung, neuroblastoma, small cell carcinoma of the lung, breast cancer, colon cancer, ovarian cancer, bladder cancer and the like.

The dose administered depends on the type of neoplastic disease, the type of patient, his age, health and weight, the type of concurrent treatment, if any, the frequency of treatment and the therapy ratio.

Dosages for the active ingredients are for example: intravenous 0.1 to about 20 mg/kg; intramuscular 1 to about 50 mg/kg; oral 5 to about 100 mg/kg; intranasal instillation 5 to about 100 mg/kg and as aerosol 5 to about 100 mg/kg. In the present case mg/kg means the weight of active ingredient in mg divided by the patient's body weight in kg.

Expressed as a concentration, the active ingredient may be present in the preparations according to the invention for topical use on the skin, for intranasal use, for use in the throat/nose area, for bronchial, intravaginal or rectal use or for use in the eye area in an amount of from about 0.01 to about 50% w/g of the preparation, for parenteral use in an amount of from about 0.05 to about 50% w/v of the preparation, preferably from about 5 to about 20% w/v.

The preparations of the invention are preferably provided for administration to humans or animals in dosage unit forms such as tablets, capsules, pills, powders, granules, suppositories, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or suspensions and oral solutions or suspensions and the like, all of which contain suitable amounts of active ingredient.

Either solid or flowable dosage units can be prepared for oral administration.

Powder is most conveniently obtained by grinding the active ingredient to a suitable fine size and mixing it with a similarly ground diluent. The diluent may consist of an edible carbohydrate material, e.g. lactose or starch. A sweetener or sugar and a flavoring oil are conveniently used.

Capsules are obtained by preparing a powder mixture in the manner described and filling it into molded gelatin shells. It is advisable to add a lubricant, such as talc, magnesium stearate, calcium stearate and the like, to the powder mixture before filling as an aid for the filling process.

Soft gelatin capsules are obtained by mechanically encapsulating a slurry of the active ingredients with an acceptable vegetable oil, light liquid petrolatum or other inert oil or triglyceride.

Tablets are obtained by providing a powder mixture as described above, granulating or crushing it, adding a lubricant and compressing the whole into tablets. The powder mixture is obtained by mixing an active ingredient which has been suitably ground with a diluent or base such as starch, lactose, kaolin, dicalcium phosphate and the like. The Powder mixture can be granulated by moistening with a binder such as corn syrup, gelatin solution, methylcellulose solution or acacia mucilage and forcing through a sieve. As an alternative to granulation, the powder mixture can also be crushed, for example by running it through the tabletting device, whereupon the resulting incompletely formed tablets are broken into pieces (blanks). The blanks can then be "lubricated" by adding stearic acid, a stearic acid salt, talc or a mineral oil to prevent sticking to the tabletting tools. The lubricated mixture is then pressed into tablets.

If desired, the tablet is provided with a protective coating in the form of a sealing coating, or with an enteric coating of shellac, a coating of sugar and methylcellulose or a polishing coating of carnauba wax.

Flowable dosage units for oral administration, such as syrups, elixirs and suspensions, can be prepared so that each teaspoonful of preparation contains a given amount of active ingredient for administration. The water-soluble forms can be dissolved in an aqueous vehicle together with sugar, flavorings and preservatives to form a syrup. An elixir is prepared using an aqueous-alcoholic vehicle with suitable sweetening agents together with a flavoring agent. Suspensions are prepared from the insoluble forms with a suitable vehicle using a suspending agent such as acacia, tragacanth, methylcellulose and the like.

For parenteral administration, flowable dosage units can be prepared using an active ingredient and a sterile carrier, preferably water. The active ingredient can be either suspended or dissolved in the carrier, depending on the form and concentration used. When preparing In the case of solutions, the water-soluble active ingredient may be dissolved in water for injection and sterilized by filtration before filling into a suitable vial or ampoule and sealing the same. Excipients such as local anaesthetics, preservatives and buffers may conveniently be dissolved in the vehicle. Parenteral suspensions are prepared in substantially the same manner, with an active ingredient suspended in the vehicle rather than dissolved in it. In this case sterilization cannot be effected by filtration. The active ingredient may be sterilized by exposure to ethylene oxide before suspension in the sterile vehicle. A surfactant or wetting agent is conveniently incorporated into the preparation to facilitate uniform distribution of the active ingredient in the vehicle.

In addition to oral and parenteral administration, rectal and vaginal routes can also be used as an effective delivery system. An active ingredient can be administered using a suppository. A suitable carrier is one with a pour point around body temperature or one that is readily soluble. Examples of suitable carriers are cocoa butter and various polyethylene glycols (carbohydrates).

For intranasal instillation, flowable dosage units are prepared with an active ingredient and a suitable pharmaceutical carrier, preferably aseptic (PE) water. If insufflation is the route of administration of choice, a dry powder can also be formulated.

For use as an aerosol, the active ingredient may be combined with a gaseous or liquid propellant, for example dichlorodifluoromethane, carbon dioxide, nitrogen, propane and the like, (as required or desired) with conventional adjuvants such as cosolvents and wetting agents, in a pressurized aerosol container.

The term "unit dosage" as used in the specification and claims refers to physically distinct units that can be administered as unit doses to humans and animals. Each unit contains a given amount of active material in an amount calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosages of the invention are dictated by and are directly dependent on (a) the unique properties of the active material and the specific therapeutic effect sought and (b) the limitations known to those skilled in the art in formulating an active material for the therapeutic use in humans discussed in this specification. Examples of suitable dosage units according to the invention are tablets, capsules, lozenges, suppositories, powder packets, wafers, wafer capsules, teaspoonfuls, tablespoonfuls, dropfuls, ampoules, vials, subdivided multiples of any of the above-mentioned dosage forms and other described dosage forms.

The active ingredients to be used as antineoplastic agents can be readily provided in such dosage units using pharmaceutical materials that are available or can be prepared by accepted methods. The following preparations illustrate the preparation of dosage units according to the invention, but are not intended to be limiting.

example 1

Using the present invention, various dosage units were prepared. They are illustrated in the following examples. The term "active "Ingredient" stands for dolastatin 13, the related dehydrodolastatin 13, a synthetic counterpart and their non-toxic, pharmaceutically active derivatives.

Preparation "A" Hard gelatin capsules

From the following types and quantities of ingredients

active ingredient, micronized 20 g

Grain starch 20 g

Talc 20 g

Magnesium stearate 2g

One thousand double-piece hard gelatin capsules for oral use, each containing 20 mg of active ingredient, are produced.

For this purpose, the active ingredient, after finely dividing it with the aid of an air micronizer, is added to the other finely powdered ingredients, after which the whole is thoroughly mixed and then encapsulated in the usual known manner.

The capsules obtained are suitable for the treatment of a neoplastic disease by oral intake of one or two capsules one to four times per day.

Following the steps described, capsules containing 5, 25 and 50 mg of active ingredient can be produced in a similar manner by replacing the initial amount of 20 g of active ingredient with 5, 25 or 50 g of active ingredient.

Preparation "B" Soft gelatin capsules

Single-piece soft gelatin capsules for oral administration are manufactured, each containing 20 mg of active ingredient (finely dispersed using an air micronizer). To make the material encapsulable, the compound in question is first suspended in 0.5 ml of corn oil, which is then encapsulated in the manner described.

The capsules obtained are suitable for the treatment of a neoplastic disease by oral intake of one or two capsules one to four times per day.

Preparation "C" Tablets

From the following types and amounts of ingredients:

active ingredient, micronized 20 g

Lactose 300g

Corn starch 50 g

Magnesium stearate 4g

light liquid Vaseline 5 g

One thousand tablets are produced, each containing 20 mg of active ingredient.

For this purpose, the active ingredient, finely divided with the aid of an air micronizer, is added to the other ingredients, after which the whole is thoroughly mixed and broken up. The blanks (obtained in this way) are further crushed by pressing them through a No. 16 sieve. The granules thus obtained are then pressed into tablets, each containing 20 mg of the active ingredient.

The tablets obtained are suitable for the treatment of a neoplastic disease by oral intake of one or two tablets one to four times a day.

In the manner described, tablets containing 25 mg or 10 mg of active ingredient can be produced by replacing the 20 g of active ingredient with 25 g or 10 g of active ingredient.

Preparation "D" Oral suspension

From the following types and amounts of ingredients:

active ingredient, micronized 1 g

Citric acid 2g

Benzoic acid 1g

Sucrose 790 g

Tragacanth 5g

Lemon oil 2g

filled with deionized water to 1000 ml

1000 ml of an aqueous suspension for oral administration is prepared with 5 mg of active ingredient per teaspoonful (5 ml).

For this purpose, the citric acid, benzoic acid, sucrose, tragacanth and lemon oil are dispersed in enough water to obtain 850 ml of suspension. The active ingredient, which has been finely divided using an air micronizer, is then stirred into the syrup until it is evenly distributed. Finally, the syrup is made up to 1000 ml with a sufficient amount of water.

The preparation is suitable for the treatment of neoplastic disease in a dose of one tablespoon (15 ml) three times a day.

Preparation "E" Parenteral product

From the following types and amounts of ingredients:

active ingredient, micronized 30 g

Polysorbate 80 5 g

Methylparaben 2.5g

Propylparaben 0.17g

filled with water for injection to 1000 ml

A sterile aqueous suspension for parenteral injection containing 30 mg of active ingredient per 1 ml is prepared for the treatment of neoplastic disease.

For this purpose, all components except the active ingredient are dissolved in water, after which the solution is sterilized by filtration. The sterile The sterilized active ingredient, finely divided using an air micronizer, is added to the solution, after which the finished suspension is filled into sterile vials and sealed.

The preparation is suitable for the treatment of neoplastic disease in a dose of 1 ml (1 M) three times a day.

Preparation "F" Suppositories for rectal and vaginal use

From the following types and amounts of ingredients:

active ingredient, micronized 1.5 g

Propylene glycol 150 g

With polyethylene glycol #4000, a thousand suppositories weighing 2.5 g each, each containing 20 mg of active ingredient, are prepared to 1,500 g.

For this purpose, the active ingredient is first finely divided using an air micronizer, then added to the propylene glycol and finally the resulting mixture is passed through a colloid mill to achieve uniform dispersion. After the polyethylene glycol has melted, the propylene glycol dispersion is slowly added to it while stirring. The suspension is filled into non-refrigerated molds at 40ºC. After the preparation has been allowed to cool and solidify, it is removed from the mold in suppository form. Each suppository is wrapped in foil.

The resulting suppositories are inserted rectally or vaginally for the treatment of neoplastic disease.

Preparation "G" Intranasal suspension

From the following types and amounts of ingredients:

active ingredient, micronized 1.5 g

Polysorbate 80 5 g

Methylparaben 2.5g

Propylparaben 0.17g

1000 ml of a sterile aqueous suspension for intranasal instillation are prepared containing 20 mg of an active ingredient per ml of suspension.

For this purpose, all the components, with the exception of the active ingredient, are dissolved in water, and the resulting solution is then sterilized by filtration. The active ingredient, which has been finely divided and sterilized using an air micronizer, is then added to the sterile solution, and the finished suspension is then aseptically filled into sterile containers.

The resulting preparation is suitable for the treatment of neoplastic disease by intranasal instillation of 0.2 to 0.5 ml one to four times a day.

An active ingredient may also be present in undiluted pure form for topical use on the cutis, for intranasal use, for use in the throat/nose/pharynx area, or for bronchial or oral use.

Preparation "H" powder

5 g of active ingredient in the form of a mass are finely divided using an air micronizer. The micronized powder is filled into a shaking container.

This preparation is suitable for the local treatment of a neoplastic disease by applying the powder one to four times per day.

Preparation "I" Oral powder

10 g of active ingredient in the form of a mass are finely divided using an air micronizer. The micronized powder is divided into individual doses of 20 mg and packaged.

The powders obtained are suitable for the treatment of a neoplastic disease by oral intake of a suspension of one or two amounts of powder in a glass of water one to four times a day.

Preparation "J" Blowing in

10 g of an active ingredient in the form of a mass are finely divided using an air micronizer.

The resulting preparation is suitable for treatment of neoplastic disease by inhalation of 30 mg one to four times a day.

Preparation "K" Hard gelatin capsules

One hundred double-piece hard gelatin capsules for oral administration, each containing 20 mg of active ingredient, are prepared.

For this purpose, the active ingredient is finely divided using an air micronizer and encapsulated in the usual manner.

The capsules obtained are suitable for the treatment of a neoplastic disease by oral intake of one or two capsules one to four times per day.

In the manner described, by replacing the 20 g of active ingredient with 5, 25 or 50 g of active ingredient, capsules containing 5, 25 or 50 mg of active ingredient can be produced.

Example 2

According to protocol 1,200 in "Cancer Chemotherapy Reports" Part 3, Volume 3, No. 2, September 1972, pages 9 ff, dosage units of dolastatin 13 provided in the selected preparation form according to Example 1 are tested for lymphocytic leukemia P388. They yielded positive results. Dolastatin 13 also caused a significant inhibition of the growth of P388 in an in vitro cell line (ED₅₀ = 1.3 x 10⊃min;² ug/ml)

The foregoing explanations clearly show that new and valuable linear depsipeptides which inhibit cell growth, as well as new and valuable cytostatic preparations which are able to solve the problems outlined at the outset in a highly unexpected manner, have been described and illustrated. Naturally, modifications, variations and adaptations which are immediately apparent to the person skilled in the art familiar with this publication also fall within the scope of the inventive concept. This is limited only by the scope of the appended claims.

Claims (1)

1. Compound structural formula: where R₁ = OH and R₂ = H. where R₁ = R₂ - Δ14.15 3. A pharmaceutical preparation comprising a pharmaceutically acceptable carrier and a compound according to claim 1 or 2. 4. A compound according to claim 1 or 2 for use in medicine. 5. A compound according to claim 4, which is administered intravenously in a dose of 0.1 up to about 20 mg per kg of patient body weight. 6. A compound according to claim 4, which is administered subcutaneously in a dose of about 1 up to about 50 mg per kg of patient body weight. 7. A compound according to claim 4, which is administered orally in a dose of about 5 up to about 100 mg per kg of patient body weight. 8. Use of a compound according to claim 1 or 2 for the manufacture of a medicament for the treatment of humans or other animals affected by a neoplastic disease. 9. Use according to claim 8, wherein the neoplastic disease is lymphocytic leukemia. 10. Use according to claim 9, wherein the amount of the compound administered is about 1 to about 4 mg per kg of patient body weight. 11. A process for preparing a compound of a formula according to claim 1 or 2 by extraction from Dolabella auricularia. 12. A process according to claim 11, comprising one or more of solvent extraction, partition chromatography, silica gel chromatography, preparative thin layer chromatography and crystallization from solvents.